Studies have demonstrated that packaging of recombinant adeno-associated virus 2 (rAAV) as self-complementary duplex strand (sc) results in early transgene expression, possibly eliminating rate-limiting second-strand synthesis. In the present study, we evaluated the molecular organization, stability of the sc AAV genome, and transgene expression in the quadriceps muscle of C57BL/6J mice in vivo as compared with single-stranded (ss) AAV. Studies were carried out with rAAV encoding green fluorescent protein (GFP) or human carcinoembryonic antigen (CEA) either as single-stranded or self-complementary duplex strand structures, encapsidated in AAV-2 capsids. Mice were injected with 1011 particles of the respective viruses and the vector-injected muscles were harvested 1 week, 2 weeks, 3 weeks, or 2 months later. Tissues were processed for total DNA isolation for the analyses of vector genomic configuration and copy number, and for immunostaining of transgene expression. ELISA was done on serum samples to quantitate CEA-specific humoral immune response as a correlate of transgene expression. Results of Southern blot and PCR analyses indicated more disintegration of the monomeric ss AAV DNA in vivo compared with linear sc AAV DNA. The results also indicated efficient conversion of the self-complementary duplex-stranded vector genome to dimer during early time points. As expected, transgene expression was detected at early time points with self-complementary duplex-stranded vector and persisted stably. However, the advantage of higher transgene expression from sc AAV was balanced over time by the single-stranded vector. These data demonstrate that sc AAV provides better stability for transgene structure during the initial stages of transduction and may have better utility in AAV gene therapy in situations, which mandate early transgene expression. © Mary Ann Liebert, Inc.